The present invention relates to novel polyenepolycarboxylic acids, or derivatives thereof at their carboxyl group(s) or their salts (which may hereinafter be referred to as xe2x80x9cpolyenepolycarboxylic acidsxe2x80x9d simply), anr d more specifically, to such polyenepolyearboxylic acids having superior dispersing ability which are produced by microorganisms belonging to genus Talaromyces.
Heretofore, there have been used surface active agents, macromolecular polymers and the like as dispersants for industrial purposes in a variety of fields such as pigments, coatings, cosmetic articles, coated paper, dyes, ceramics, and building and civil engineering, and particularly in recent years such compounds have been generally used in the production of fine particles.
Since these compounds thus widely used as dispersants for industrial purposes are frequently synthetic organic compounds, such compounds mostly are inferior in safety, biodegradability and like characteristics though possessing excellent dispersing ability. Therefore, depending on the manner of use, there is a fear that such compounds may raise problems such as bad influence on living bodies, environmental pollution, and scattering of such compounds in the environment.
Attention is now focused on dispersants originating from natural products which exhibit superiority in both the biodegradability and safety and hence are friendly to the terrestrial environment. Examples of such dispersants include xanthan gum, which is a polysaccharide produced by a microorganism and which is widely used in various industrial fields, taking advantage of its viscosity. However, xanthan gum has poor dispersing ability with respect to inorganic or oleaginous ingredients used in pigments, coatings and the like though offering superior safety.
Other such dispersants include saccharic acids such as sodium gluconate, which are used in a part of industry but find a limited range of application as dispersants since situations and sorts of dispersoids to which they are applicable are limited.
Accordingly, it has been earnestly desired to provide a compound originating from a natural product which is capable of exhibiting superior dispersing ability irrespective of the sort of dispersoid be used.
A compound according to the present invention having attained the above object is characterized by polyenepolycarboxylic acids represented by the following formula (1): 
(wherein n represents an integer of 0 to 5 and m represents an integer of 1 or 2, provided m is 1 when n is 0), or derivatives thereof at their carboxyl group(s) or their salts. Those compounds of the above formula (1) wherein m=1 and n=0; m=2 and n=1; m=2 and n=2 are preferred embodiments of the present invention.
A dispersant according to the present invention having attained the above object is characterized by containing polyenepolycarboxylic acid(s) represented by the above formula (1) wherein n represents an integer of 0 to 5, and m represents an integer of 1 or 2, or derivatives thereof at their carboxyl group(s), or their salts. Any of those compounds of the above formula (1) wherein m=2 and n=0; m 2 and n=1; m=2 and n=2 is a preferred embodiment of the present invention.
The polyenepolycarboxylic acids represented by the above formula (1) (wherein n represents an integer of 0 to 5 and m represents an integer of 1 or 2), or acid antydrides thereof or their s alts are prepared by the use of a culture solution obtained by cultivating a microorganism belonging to genus Talaromyces in a culture medium dand hence, such a preparation method is included in the scope of the invention. Also, the microorganisms that produce the polyenepolycarboxylic acids represented by the above formula (1) (wherein n rep resents an integer of 0 to 5 and m represents an integer of 1 or 2), or derivatives thereof at its carboxyl group(s) or their salts are included in the scope of the invention.
It is to be noted that for convenience the polyenepolycarboxylic acids represented by the above formula (1) (wherein n represents an integer of 0 to 5 and m represents an integer of 1 or 2), or derivatives thereof at their carboxyl group(s) or their salts will sometimes be referred to as xe2x80x9cthe compounds of the present inventionxe2x80x9d in the following description. Further, for simplification, the polyenepolycarboxylic acid of the above formula (1) wherein m=1 and n=0 will be referred to as xe2x80x9cR0 compoundxe2x80x9d; the polyenepolycarboxylic acid of the above formula (1) wherein m=2 and n=0 as xe2x80x9cS0 compoundsxe2x80x9d; the polyenepolycarboxylic acid of the above formula (1) wherein m=2 and n=1 as xe2x80x9cS1 compoundxe2x80x9d; and the polyenepolycarboxylic acid of the above formula (1) wherein m=2 and n=2 as xe2x80x9cS2 compoundxe2x80x9d. Similarly, those R0, S0, S1 and S2 compounds in which two carboxyl groups bonded to adjacent carbon atoms are entirely bonded together to form an acid anhydride will simply be referred to as xe2x80x9cR0 anhydridexe2x80x9d, xe2x80x9cS0 anhydridexe2x80x9d, xe2x80x9cS1 anhyd ridexe2x80x9d, and xe2x80x9cS2 anhydridexe2x80x9d, respectively. The structural formulae of these compounds are shown below. 
As a result of intensive study by the inventors of the present invention, it has been discovered that compounds produced by strain No. 10092 belonging to genus Talaromyces have superior dispersing ability with respect to various dispersoids in whether aqueous or non-aqueous systems. The present invention has been made based on this discovery.
The compounds represented by the above formula (1) wherein n is an integer of 1 to 5 and m is 1 or 2, or wherein n is 0 and m is 1 are novel compounds that were discovered by the inventors of the present invention for the first time. It should be noted that the compound of the formula (1) wherein n is 0 and m is 2 is known from, for example, Aldridge, D C et al, J. C. S. Perkin I, 1980, 2134 (isol, struet, nmr), which discloses an acid anhydride of a tricarboxylic acid represented by the following formula: 
and describes that this compound is obtainable from Paecilomyces variotii, a sort of fungi. Another literature reference, Jabbar, A et al., Pharmazie, 1995, 50, 706 (isol, props), describes that an analogous compound can be isolated by Penicillium sp. These literature references, however, are silent on the dispersing ability of the aforementioned compounds.
Accordingly, the compounds of the formula (1) wherein n is an integer of 0 to 5 and m is 1 or 2 are within the scope of the present invention, so long as they are used as dispersants.
In the description of the instant application, the term xe2x80x9clowerxe2x80x9d means the number of carbon atoms from 1 to 6.
The compounds of the present invention include, in addition to the polyenepolycarboxylic acids represented by the formula (1), derivatives thereof at its carboxyl group(s) or their salts.
Examples of xe2x80x9cderivatives thereof at their carboxyl groupsxe2x80x9d include compounds of the formula (1) wherein the carboxyl groups thereof are, either partially or entirely, salts, anhydrides, esters, amides or the like. These derivatives also include compounds of the formula (1) wherein the carboxyl groups thereof are, either partially or entirely, in combination of them. Specifically, examples of such derivatives include compounds of the formula (1) wherein part of the carboxyl groups is an ester, while the rest is an anhydride. The above expression xe2x80x9ctheir saltsxe2x80x9d means salts of the for egoing derivatives, examples of which include compounds of the formula (1) wherein part of the carboxyl groups is a derivative as above, while the rest is a salt.
The xe2x80x9csaltsxe2x80x9d as stated above are any common non-toxic salts without any particular limitation to specific sorts thereof. Examples of such salts include alkali metal salts (such as sodium salts and potassium salts), alkali earth metal salts (such as calcium salts and magnesium salts), ammonium; salts, organic base salts (such as trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salts and N,Nxe2x80x2-dibenzylethylenediamine salts), and salts with amino acids (such as arginine salts, asparates and glutamates).
Examples of the xe2x80x9canhydridesxe2x80x9d as stated above include those in which two carboxyl groups bonded to adjacent carbon atoms are combined into an acid anhydride group. An example of such an anhydride is shown below. 
The xe2x80x9cestersxe2x80x9d as stated above include esterified carboxyl groups, and examples of preferred esters include alkyl esters including lower alkyl esters such as inethyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, tertiary butyl ester, pentyl ester and hexyl ester.
The xe2x80x9camidesxe2x80x9d as stated above include amidated carboxyl groups, which may be condensed like a maleimide. Examples of such amides include lower alkylamides such as monomethylamide and monoethylamide; and arylamides such as monophenylamide, monobenzylamide and phenetylamide.
The xe2x80x9csaltsxe2x80x9d, xe2x80x9canhydridesxe2x80x9d, xe2x80x9cestersxe2x80x9d and xe2x80x9camidesxe2x80x9d described above may be prepared according to conventional methods.
The above xe2x80x9canhydridesxe2x80x9d each may be prepared, for example, by a method such as to subject the polyenepolycarboxylic acids of the formula (1) to a dehydration reaction.
The above xe2x80x9cestersxe2x80x9d each may be prepared by a method such as to react the polyonopolycarboxylic acids of the formula (1) with an alcohol or as to subject acid anhydrides of the polyenepolycarboxylic acids to esterification reaction.
The above xe2x80x9camidesxe2x80x9d each may be prepared by a method such as to subject ammonium salts of the polyenepolyearboxylic acids represented by the formula (1) to a dehydration reaction, or to subject nitrile compounds of the polyenepolycarb oxylic acids to a saponification reaction, or to subject acid anhydrides or esters of the polyenepolycarboxylic acids or the Like to amidation reaction.
The S1 and S2 compounds of the foregoing compounds of the present invention have been confirmed to be extremely low toxic and safe compounds since they showed negativity in a mutagenicity test and no particular abnormality in a mouse acute toxicity test (oral administration: 2.0 g/kg).
Next, methods of obtaining the compounds of the present invention from a microorganism belonging to genus Talaromyces are described below.
First, a method of cultivating the microorganism is described. This microorganism, the characteristics of which will be described later, belongs to fungi, or what is commonly called xe2x80x9cmoldxe2x80x9d (filamentous fungi) and, hence, generally known culture media for fungi may be used in cultivating the microorganism. Examples of such culture media include those comprising a carbon source, a nitrogen source, inorganic salts and a trace nutrient source. As the carbon source can be used glucose, xylose, fructose, sucrose, maltose, starch, oxidized starch, and hydrolyzed starch. As the nitrogen source can be used natural products such as corn steep liquor; inorganic nitrogen compounds such as sodium nitrate; and amino acids such as leucine and lysine. As the inorganic salts can be used calcium carbonate and magnesium sulfate or the like; and as the trace elements can be used iron sulfate or the like.
A culture medium to be used may be either a liquid medium or a solid medium, which is preferably subjected to shaking culture or aeration spinner culture. If the pH and temperature of such a culture medium under cultivation are adjusted to 3-8 and to 20-40xc2x0 C., respectively, it is possible to obtain an effectively active components with higher productivity. It is recommended that the incubation period be three to five days, and a culture may. be obtained by a continuous fermentation as well as by a batch process.
The compounds of the present invention are obtained by extracting the culture thus obtained, followed by isolation or purification. Specifically, the culture obtained by the above method is separated into the culture solution and the fungal mass by filtration or centrifugal separation to afford a culture filtrate containing the compounds of the present invention. The culture filtrate is subjected to conventional extraction and isolation or purification processes, thus giving the compounds of the present invention. These extraction and isolation or purification processes are not particularly limited and can be appropriately selected from conventional processes. To isolate or purify the compounds of the present invention, for example, it is possible to adopt processes such as an acid treatment in which an acid such as a mineral acid or an organic acid is added to the culture filtrate, a treatment using an ion exchange resin, an adsorbing resin or the like, and a process using a dialysis membrane, an ultrafiltration membrane or gel permeation.
The compounds thus obtained are each treated by conventional methods to give derivatives thereof at their carboxyl group(s) or their salts. Derivatives, for example, esters or amides, of such a compound of the present invention can be obtained by subjecting a crude product resulting from concentration and evaporation to dryness of the aforementioned filtrate to a chemical reaction to give the derivatives such as esters or amides, followed by purification according to a conventional purification method adapted for fat-soluble substances. These derivatives may be hydrolyzed to give the compounds of the present invention and salts thereof. As to a series of these processes, reference may be made to methods described in examples to be described later.
The compounds (1), or derivatives thereof at their carboxyl group(s) or their salts thus obtained exhibit superior dispersing ability with respect to a wide range of organic and inorganic dispersoids in aqueous or non-aqueous systems and hence can be used as dispersants in various industrial fields such as coatings, pigments, inks, fiber, paper, cosmetic articles, foods, contrast media, paints, cement, concrete, rubber, plastics, ink jet, pharmaceuticals, agricultural chemicals, dyes, glazes, COM [Coal Oil Mixture (finely-divided coal)], photo graphic films, magnetic tapes, scale, heavy oil, and cleaning dispersants (lubricant oil additives).
Further, the compounds of the present invention also possess a chelating action. That is, the compounds of the present invention chelate with interfering metal ions thereby masking these metal ions. By making use of such an action, the compounds can be used as additives to detergents or dyes, and as stabilizers for foods, chemicals, cosmetic articles, and the like. Specifically, the compounds of the present invenion can find use in broad industrial fields such as metallic surface cleaning agents, bottle washing agents used in the food processing industry, glass instrument washing agents used in hospitals and like facilities, and dye-assist agents used in the dyeing industry.
Furthermore, the compounds of the present invention also possess a surface activity. By making use of such an activity, the compounds can be widely used in detergents for clothes, kitchen, hair, face and body, in emulsifying agents for cosmetic articles and agricultural chemicals, in anti static agents for clothes, and in like applications.
As to the amount of the dispersant of the present invention, a suitable amount range can appropriately be selected depending on the sort of dispersoid to be used or a like factor. Generally, addition of the dispersant in a range from 0.01 to 20 wt % to a dispersoid is preferable. The addition of the dispersant in an amount of less than 0.01 wt % would not result in a desired dispersing action. The addition of not less than 0.1 wt % of the dispersant is more preferable. On the other hand, the addition of more than 20 wt % of the dispersant would raise problems such as an occurrence of precipitation. The addition of not more than 10 wt % is more preferable.
Description will follow of microorganisms belonging to genus Talaromyces that can produce the compounds of the present invention. It should be noted that the following description is directed to the characteristics of strain No. 10092 of filamentous fungi isolated by the inventors of the instant application as a representative example but never intends to limit to this microorganism.
The above strain No. 10092 of filamentous fungi has been separated from soil collected at Ibusuki city, Kagoshima, Japan. This strain can widely grow on various culture media and form a colony with a color tone from yellowish white to light yellow. The strain formed many teleomorphg (ascocarps) and a few anamorphs (conidium structures) on a culture medium. The teleomorphs were each a spherical ascocarp in an orange color covered with undifferentiated hyphae in which asci lay scattered. The ascospore thereof was a colorless, single cell, wide elliptic and was observed to have a ridge on an equatorial surface. On the other hand, the anamorphs each comprised a single conidiogenous cell or broom-like conidiophore and formed chain-linked conidia each shaped spherical and comprised of a single cell. The mycological characteristics of the strain are as follows.
The attributes of cultures resulting on various agar culture media are summarized in Tables 1 to 3.
It should be noted that the data in Table 1 were those observed after incubation at 25xc2x0 C. for seven days from inoculation. The color tones were described based on Me thuen Handbook of Colour (Komerup, A. and J. H. Wanscher, 3rd ed., 525p., Methuen, London, 1978).
Further, the data in Tables 2 and 3 were those observed after incubation at 25xc2x0 C. for 14 days from inoculation. The color tones were observed and determined in the same manner as in Table 1.
From these Tables, considerations can be given as follows.
The foregoing strain was rapidly cultivated in the yeast extract-added Czapek agar culture medium, and after incubation at 25xc2x0 C. for seven days, it formed a circular colony having a diameter of 4.7 to 5.2 cm. The face of this colony was flat and felt-like, and formed with radial grooves. The color tone of the colony was white or yellowish white to light yellow, and that of the reverse face of the colony was yellowish white or grayish yellow. Neither teleomorph nor anamorph was observed.
In the case of cultivation in the malt-extracted agar culture medium, a colony was obtained more rapidly than in the above culture medium, and the size of the colony cultivated under the same condition as above reached 6.2 to 7.0 cm in diameter. This colony expanded circularly with its face being flat, in a felt-like to granular state and in a color tone from white or yellowish white to light yellow. The reverse face of the colony exhibited a color tone from yellow to grayish yellow and diffused a soluble yellow dye in the culture medium. On this culture medium was formed a small quantity of anamorphs. When the cultivation in the malt-extracted agar culture medium was conducted for two weeks, the resulting colony expanded to have a diameter of 8 cm or more, as noted in Table 2, and reached a wall surface of the Petri dish. The face of this colony was flat and in a felt-like to granular state, and formed a small quantity of anamorphs as well as abundance of teleomorphs. The colony exhibited a color tone from yellowish white to light yellow, and the reverse face thereof exhibited a color tone from yellow to grayish yellow and diffused a soluble yellow dye in the culture medium.
The morphological characteristics of the strain were determined based on the results of culture on an LCA agar culture medium (Miura culture medium) developed by Miura and Kudo (Miura, K. and M. Kudo: Trans. Mycol. Soc. Japan, 11: 116-118, 1970). As a result, there were observed abundant teleomorphs (ascocarps) and anamorphs (conidium structures).
These ascocarps were superficially present and independent, each shaped spherical to subspherical with a diameter of 350 xcexcm at the maximum, and of the non-ostiolate form. The surface of each ascocarp was covered with interwoven hypha network and exhibited a color tone from yellowish white to orange. Within each ascocarp were scattered asci which were each evanescent, subspherical to elliptic, and 7 to 10 xcexcm in diameter, and which each had eight ascospores therein. These ascospores exhibited a color tone from colorless to light orange, and each had a somewhat coarse surface, a single cell, and a wide elliptic to lens shape. Each of the ascospores further had a single ridge on an equatorial surface and a size of 3 to 4.5 xcexcmxc3x972.5 to 3 xcexcm.
On the other hand, the conidium structure was of the phialo type in which conidia were formed as linked from phialides. These phialide8 generated as branching of aerial or substrate mycelia, or three to five such phialides were formed at the tip of a broomlike conidiophore. They were colorless and had smooth surfaces, pen-tip shapes, and a size of 13 to 18 xcexcmxc3x971.5 to 3.5 xcexcm as a mononematous or 8 to 14 xcexcmxc3x971.5 to 3.5 xcexcm as a verticillate. Each of the conidia was colorless an d had a smooth surface, a single cell, a spherical to wide elliptic or oval shape, and a size of 2 to 4 (or to 5) xcexcmxc3x972 to 3.5 xcexcm. A vegetative hypha had a smooth surface and a septal wall, and was colorless and branching. The hypha cell was cylindrical and 1.5 to 6.5 xcexcm wide, but any chlamydospore was not formed.
Examination of the foregoing strain as to its optimum temperature for growth with use of a potato dextrose agar culture medium (available from NISSUI SEIYAKU) proved that the strain could grow within a temperature range between 7xc2x0 C. and 40xc2x0 C., and that its optimum temperature for growth was 29xc2x0 to 34xc2x0 C.
When the foregoing mycological characteristics were compared to the fungi classification standard by von Arx (J. A. von Arx: The Genera of Fungi-Sporulating in Pure Culture. 3rd ed., J. Cramer, Vaduz, 1974) and the monograph by Pitt (Pitt, J. I., The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces, Academic Press, London, 1979), the strain No. 10092 was considered to belong to genus Talaromyces (Talaromyces C. R. Benji. 1955) of Plectomycetes. Thus, this strain was identified as one strain of genus Talaromyces and named xe2x80x9cTalaromyces sp. No. 10092xe2x80x9d. It should be noted that this strain has been deposited to National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology as an International Depositary Authority established under the Budapest Treaty, with a deposit No. FERM BP-6250 (Date of acceptance of deposit: Feb. 9, 1998).